The N-end rule relates the in vivo half-life of a protein to the identity of its amino-terminal residue. Overexpression of Ubr1p and Ubc2p, the targeting components of the N-end rule pathway, greatly impairs viability and growth. This toxicity was traced to the enhanced degradation of G- alpha, a subunit of the heterotrimeric G-protein which regulates mating in yeast. A study of G-alpha degradation may make important contributions to understanding the significance of ubiquitin-dependent proteolysis during signal transduction. Four goals are described to explore the mechanism of G-alpha degradation. 1) The degradation signal on G-alpha that allows it to be recognized by the N-end rule pathway will be sought. To identify a G-alpha-specific degradation signal, truncations of G-alpha will be constructed and the stability of the mutants will be investigated. Reporter genes, encoding G- alpha-beta-gal and G-alpha-Ura3, will be used to isolate intra- and extragenic mutants that affect G-alpha stability. 2) The site of ubiquitin attachment will be investigated. Ubiquitinated G- alpha will be chemically cleaved and the products resolved on two- dimensional gels to identify site(s) of ubiquitin conjugation. G-alpha mutants constructed in Aim l above will be tested for their ability to support G-alpha ubiquitination. The phenotype and stability of G-alpha mutants that cannot be ubiquitinated will be assessed. 3) The possibility that G-alpha degradation might be promoted in trans will be investigated. We will concentrate on Sst2, a protein required for degradation of G-alpha and for desensitizing cells to chronic pheromone response. The stability of G-alpha in various SST2 backgrounds will be tested. Epitope-tagged versions of G-alpha and Sst2p will be constructed, to test if these two proteins interact and truncated derivatives of G- alpha (from Aim l above), will be used to map the interaction domain. 4) The effect of post-translational modifications on G-alpha stability will be explored. We will study the effect on G-alpha stability, of membrane binding, ubiquitination, GTP-hydrolysis, and treatment with mating-pheromone. In mammalian cells perturbations in the G-protein signaling pathway are implicated in many diseases, including hyperthyroidism and precocious puberty. Defects in the ubiquitin pathway have been implicated in Alzheimer's disease and in the growth defect of a mouse cell-line, ts85. The evolutionary conservation of the ubiquitin and the hormone-induced signal transduction pathways, suggest that advances from this work might be broadly applicable to other systems. Indeed, Gi-alpha and Ubc2p from mammals can functionally replace their counterparts in yeast. It is anticipated that a study of G-alpha degradation, the first physiologically relevant substrate of this pathway, will be important to both basic and clinical fields.